isolation characterization indole-3-acetic acid from ... · reagent to visualize indoles...

Plant Physiol. (1987) 84, 1107-11130032-0889/87/84/1107/07/$0 1.00/0

Isolation and Characterization of Esters of Indole-3-AceticAcid from the Liquid Endosperm of the Horse Chestnut(Aesculus species)1

Received for publication October 21, 1986 and in revised form March 17, 1987

WOJCIECH DOMAGALSKI2, AGA SCHULZE, AND ROBERT S. BANDURSKI*Department ofBotany and Plant Pathology, Michigan State University, East Lansing, Michigan 48824

ABSTRACT

Esters of indole-3-acetic acid were extracted and purified from theliquid endosperm of immature fruits of various species of the horsechestnut (Aesculus parvipiora, A. baumanni, A.pavia rubra, and A. paviahumulis). The liquid endosperm contained, at least 12 chromatographi-cally distinct esters. One of these compounds was purified and charac-terized as an ester of indole-3-acetic acid and myo-inositol. A secondcompound was found to be an ester of indole-3-acetic acid and thedisaccharide rutinose (glucosyl-rhamnose). A third compound was par-tially characterized as an ester of indole-3-acetic acid and a desoxyami-nohexose.

Shantz and Steward (26) demonstrated that a substance iso-lated from the vesicular embryo sac of immature fruits of Aes-culus woerlitzensis greatly stimulated cell division of carrot rootphloem explants. They purified this material to homogeneityand characterized its hydrolysis products as IAA, and a disaccha-ride composed of glucose and rhamnose. The instrumentationavailable did not permit characterization ofthe intact compound.An earlier study from FC Steward's laboratory demonstrated thatcoconut milk was a stimulatory adjunct to tissue culture media(3) and additional studies showed that the liquid endospermfrom the walnut (Juglans regia) and from immature grains ofZea mays were alternative sources of the stimulatory adjunct(26).Our studies of the indolylic compounds of kernels of Z. mays

resulted in the characterization of several esters of IAA and myo-inositol and myo-inositol glycosides (e.g. Refs. 6, 19, 27, 28).IAA-myo-inositol esters were also found to be present in othermembers of the Zea tribe, in rice (Oryza sativa) (14), and invegetative tissues of maize (4). We also examined the indolyliccompounds of horse chestnut liquid endosperm and observed anumber of esters of IAA (A Schulze, RS Bandurski, unpublisheddata, 1976).

This work, and a previously published abstract of a portion ofthis work (W Domagalski, A Schulze, RS Bandurski 1985, Plant

'This work was supported by the Metabolic Biology Section of theNational Science Foundation (NSF-PCM-8204017 and DMB-850423 1)and the Space Biology Program of the National Aeronautic and SpaceAdministration (NASA-NAGW-97) and the Flight Program (NAG 2-362).

2 On leave from: The Plant Breeding and Acclimatization Institute,Prezebedowo-7, 62-095 Morowana Goslina, Poznan, Poland.

Table I. Thin Layer Chromatographic Profile ofIAA Esters inAesculus sp

Chromatography was on Silica Gel 60 using ethylacetate:acetonitrile:ethanol:water (5:3:1:1) as solvent. Detection was with theEhmann reagent (9). Ammonolysis of these compounds under mildcondition yields IAA and IAA-amide indicating the compounds are IAAesters. Migration is expressed relative to that of the B, (1 1, 19) indole-3-acetyl-myo-inositol ester (RLAlnos) where the migration of IAInos = 1.0.

RjAInos A. parvifiora A. baumanni A. pavia humulislA. pavia rubra0.32 +0.41 + +0.57 +0.62 +0.70 +0.84 +0.87 +0.93 + + +1.00 + + +1.04 +1.07 + + +1.10 + +1.12 +1.14 + + +1.22 +1.46 +1.52 +1.61-66 + +1.88 +2.20 +

Physiol 77:S-3, abstract), showed IAA-myo-inositol (IAInos)3 tobe present in horse chestnut. Aharoni and Cohen showed thecompound to be present in IAA treated tobacco (N Aharoni, JDCohen 1986, Plant Physiol 80:S-34, abstract), and thus we knowthat IAInos is widely distributed in nature.The present studies arose from our prior interest in esters of

IAA (6, 19, 28) and from the studies of FC Steward's laboratory(3, 21, 26) indicating that endosperm factors provided valuableadjuncts to plant tissue culture media. Thus, the data presentedhere extend the taxonomic distribution of esters of IAA andmyo-inositol, increases the number ofknown esters, and providesknowledge of stimulatory adjuncts for plant tissue culture.

3 Abbreviations: 1AInos, indole-3-acetyl-myo-inositol; R>Ano, thinlayer chromatography migration relative to indole-3-acetyl-myo-inositol.FAB, fast atom bombardment; LH-20, lipophyllic Sephadex: EI, electronimpact; m+, molecular ion; m/z, mass per number of charges; TMS,trimethyl silyl.

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DOMAGALSKI ET AL. Plant Physiol. Vol. 84, 1987

100.

80

Z 1W 601- I

wZ 4040

w

20

.1,

185

165.1

15.1

149.1Quin

131

123

100

100j

80}

1-

601

zSugai

< 404c

w

20 38.

315.2 333.2

i ll

277

(2G * H)*

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260.1

241.1

315 .1 23,1.

200m/z

369

(4G H)+

ir- CH3334

Sugar349

Il SI llllIi III

I 0?.

j 331 )1

300

461

(50 *H)+

M - 2TMS,4H425

393g1 .29 4l2ll Sj 1381.3 393.2 482.3

FIG. 1. FAB spectrum of a putative IAA-desoxy-aminohexose isolated from A. parviflora. The mo-

lecular ion, M+ is at 609. Glycerol ions are desig-nated as G and shown at m/z = 645, 553, 461, 369,277, and 185. Diagnostic ions are labeled in thefigure using the minus symbol to indicate loss ofthat fragment by the parent ion.

II II.UII4II± U JUULLUUIiIUIL..LAUaiUlIIIIUlUnLIuiJfliU.LIIuIlJllhjjIlIljflUUIUUUIlIlUIUflIllj3li1.111111 I 1111U11111111111111111il1.llIjLUllluhIlThJIll

300 400m/z

500

1001

80-

0-

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M0Il- 40ot

20

(6G *H)-553

M* -TMS, 2H517

.25406. 1.I I., 531,3,3.3

500

.1

M1 (IAA-deoxyhexoseamine, 4TMS)

609 645

(7G. H)

565 2 525.3 583.3 592 2599 3 h,1. X 681.b67 619.4 63.;3

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m/z

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700

MATERIALS AND METHODS

Plant Material. Collections of fluid from the vesicular embryosac (liquid endosperm) were made during late August to earlySeptember during the summers of 1976, 1977, 1978, 1984, and1985. The fruits of Aesculus parviflora are small and fluid couldonly be obtained from vesicular sacs in which the embryo hadaborted, thus preventing the liquid endosperm from being ab-sorbed by the developing cotyledons (26). From the remaining

species it was possible to obtain about 0.05 to as much as 0.5 mlof fluid, usually 0.1 to 0.2 ml, from each immature vesicle sac.The fruits were cut approximately in half and the fluid aspiratedwith a syringe and collected into an iced beaker. After about 20ml of fluid had been collected, an equal volume of acetone was

added. The 1:1 fluid:acetone mixture then could be stored at-20°C for periods of up to 10 years without serious loss of theester conjugates. Collections were made from the Michigan State

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INDOLE-3-ACETIC ACID ESTERS FROM AESCULUS SPECIES

Table II. A Comparison ofthe 70 eVMass Spectral FragmentationPattern ofAcetylated Putative Indole-3-Acetyl-myo-Inositolfrom A.parviflora with Authentic Indole-3-Acetyl-myo-Inositolfrom Z. mays

m/z A. parviflora Z. mays

abundance589 0.1 0.2 Hexaacetyl IAInos547 5.0 4.0 Pentaacetyl IAInos374 0.7 1.4 547-174175 1.5 1.5 IAA174 3.7 4.6 IAA-H169 3.2 4.6157 82.3 83.2 IAA fragment145 1.4 2.7 Inositol fragment130 100.0 100.0 IAA quinolinium ion77 3.8 5.8 Benzene ring

University campus and from the following species, A. parviflora,A. hippocastanum, A. pavia humulis, and A. pavia rubra.

Chromatography. Dowex 50-X2-400 (Sigma) was prepared bycycling the resin through 1 M NaOH, water, 1 M HCI, water, 1 M

NaOH, and then water until the eluate was neutral. The purifiedresin was then stored in 50% ethanol-water (v/v) and washedbriefly just prior to use. The column dimensions were 0.9 x 19cm and elution was with 50% aqueous ethanol using a flow rateof 2 ml h-' collecting 1 ml fractions (10).LH-20 Chromatography. Lipophyllic Sephadex (LH-20) was

washed extensively with alcohol and water and then stored in50% ethanol water. Column dimensions were 2.0 x 23 cm andelution was with 50% aqueous ethanol using a flow rate of 2.0ml-h' and collecting fractions of 2.5 ml.Thin Layer Chromatography. TLC was on precoated plates of

Silica Gel 60 (Merck Darmstadt, Brinkman) using ethyl ace-tate:methyl ethyl ketone:ethanol:water (5:3:1:1) as solvent. In-dole compounds were detected by dipping the plates in Ehmanns'reagent, blotting dry, and heating at 100°C. Following washing,the plates may be kept indefinitely with retention of the charac-teristic blue color for indoles (9).HPLC Chromatography. A Varian model 5000 gradient liquid

chromatograph equipped with a Rheodyne high pressure loopinjector and a UV detector was used for HPLC analysis. Columndimensions were 4 x 250 mm and flow rates of 1 ml * min-' wereused. Reverse phase columns were packed with C18 Partasil 10-ODS. Straight phase chromatography was on Partasil 10. Thestraight phase solvent system used was ethyl acetate:acetonitrile:EtOH: H20 (65:21:7:7) or as indicated. ODS solventsystems are as described.Mass Spectrometry. FAB spectra were obtained with a VG-

ZAB-HF mass spectrometer (Shell Development, Modesto, CA).The sample was inserted into the probe in a glass capillary withglycerol and manually sublimed using resistance heating andscanning over the mass range 100 to 1200. El probe spectra wereobtained with that same instrument. The spectra for the hepa-taacetyl rutinose were obtained with a Hewlett-Packard 5992Ausing a 11 m CP Sil- 19 CB-WCOT, fused silica capillary column.Spectra of the IAA-myo-inositols were obtained at 70 eV usinga Hewlett-Packard 5985 quadrapole and 60 cm x 3 mm 3% OV-17, column.Chemicals. Chemicals were of the best available grade. Pyri-

dine was distilled over NaOH and stored under N2 in a desiccatorover calcium sulfate at -20°C. Dowex-50, LH-20 Sephadex,heptaacetyl rutinose were from Sigma Chemical Co.; 4-dimethylaminopyridine was from Aldrich Chem. Co. C18 Partasil 10 ODSand Partasil 10 were from Whatman, Clifton, NJ. TLC plateswere from Brinkman, Westbury, NY manufactured by Merck-Darmstadt.

RESULTS

Fractionation of Extracts. The 1:1 endosperm fluid:acetonemixture was filtered and then concentrated in vacuo to about 3%of the original volume. This was applied to a Dowex-50 columnand eluted with ethanol:water (1:1). The column eluate wasmonitored by TLC of the concentrated effluent using Ehmanns'reagent to visualize indoles including esters (9, 24). Usually thefirst 15 ml contained high concentrations of sugars and little orno indolylic compounds and was discarded. The ester fractionsemerge between 18 to 36 ml, as judged by Ehmann-reactivematerial, except for the putative amino hexose ester, whichemerged between 38 to 80 ml. The desired fractions were pooled,evaporated in vacuo to dryness, dissolved in ethanol:water andapplied to a 2 x 23 cm column of LH-20 sephadex which hadbeen equilibrated with 50% ethanol-water. Elution was with thesame solvent. The successive aliquots were again monitored byTLC and the ester fraction eluted between 55 to 75 ml. The TLCprofiles of all four species were similar and showed Ehmanns-reactive spots at R values relative to the B- 1 spot of IAlnos lAInos)as shown in Table 1.

Isolation and Partial Characterization of a Putative IAA-Aminodesoxy Hexose. An IAA ester was isolated from Aesculusparviflora which had characteristics suggestive of a desoxyami-nohexose ester of IAA. The compound was present in only traceamounts and a complete characterization has not been possible.The preparation was as follows: 50 ml of endosperm fluid fromA. parviflora was mixed with acetone and treated as described in"Materials and Methods." Esters eluted in the 18 to 36 mlfraction, but an Ehmann-reactive compound was observed inthe 38 to 80 ml fraction. This fraction is approximately wheretryptophan would elute, since an amino compound would bezwitterionic and would be slightly retained by a sulfonic acidresin. The 38 to 80 ml fractions were pooled and concentrated,and, owing to the minute amount of material available, the LH-20 column chromatography was omitted. The fraction was ap-plied to a straight phase Partasil 10 column and eluted with ethylacetate:acetonitrile:ethanol:water (50:20:15:15). With this sol-vent, the Ehmann-reactive compound eluted as a single peak at9.1 min. The peak was concentrated, applied to a C18 column,and developed with 5% ethanol:95% water. The compoundeluted at 9.5 min and the apparently homogeneous substance,was analyzed by FAB using the VG-ZAB instrument. The massspectral fragmentation pattern is shown in Figure 1. The molec-ular ion is at 609 and this corresponds to a 4 TMS-IAA-desoxy-aminohexose. Glycerol matrix ions are identified as G + H. M+minus TMS and 2 H is at 517, M minus 2 TMS and 4 H is at425 and major sugar ions are at 349 and 334. The TMS quino-linium ion, derived by cyclization and enlargement of the IAA(27) is at 202 and the quinolinium ion is shown at 131.

Isolation and Characterization of Esters of IAA and myo-Inositol. This compound was initially isolated from kernels ofZea mays (19) and in that tissue is the most abundant IAA ester.Endosperm fluid from A. parviflora, 38 ml was mixed with anequal volume of acetone, filtered, concentrated, and applied toa Dow 50 column. The eluate was monitored by TLC. Tubes 12to 31 showed distinct Ehmann-reactive material at the properposition for the IAA inositols and were pooled and concentratedto an aqueous phase and placed on an LH-20 column and elutedwith 50% ethanol. Substances with a migration corresponding tothat of the two spots of IAInos on TLC emerged at 26 and 27ml and were pooled and concentrated to dryness in vacuo. Thismaterial was dissolved in 50% ethanol and chromatographed ona C18 column using 5% ethanol-95% water as solvent and showed280 nm absorbency at 4.8, 8.5, 10.0, and 13.7 min. AuthenticIAInos gave an identical elution pattern. The peaks from theplant sample were pooled and chromatographed a second timeas above. The same peaks were collected. pooled, dried, and

1109



1110 DOMAGALSKI ET AL. Plant Physiol. Vol. 84, 1987

I-

zCI

I--4w

m/z

487.2

3G+Na+ (4G+H)+299.1 36 .2 391.3

[AAGLUCOSE MNUS OXYGEN3 72

315.1

391322 33322341.1 351.2

131. 359 .2

(5G+H)4,1.2

453.2443.2

433.2

45.2

i1 1i L ;JII Ill]' 11ii 1.llhldi1AjdaiWwLL1dd1uAtkIIHL.&kiAiimflyuuRUN300 350 400

m/z

1001M+H (IAA RUTINOSE)

5 6 M'+ Na (IAA-RUTINOSE)

80 48480-

w 602-az

210 ~~~~~~~(eG+H)+-

40 553.3

'LI

20, 21. 529.4 (6G+Nayt495.2 523.4 .3 576.2

516 2 %2 5 %3.30

550m/z

450

FIG. 2. FAB spectrum of the putative IAA-rutinose isolatedfrom A. parviflora. The molecular ion plus a proton is at 484,and the molecular ion plus sodium is at 506. IAA-glucoseminus oxygen is at 322. IAA is at 175 and the quinoliniumion of IAA is at 130.

11i

600

acetylated using 50 zd of pyridine, containing 0.5% of N-dime-thylaminopyridine (13). To this was added 50 ul of reagent gradeacetic anhydride. Acetylation was for 10 min at 25°C, althoughthe sample may be left at room temperature for several days.The sample was then dried in a stream of N2 gas and dissolvedin 25Ml of ethylacetate for GC-MS. Mass spectra were obtainedat 70 eV using a Hewlett-Packard 5985 quadrapole GC-MS witha 60 cm x 3 mm 3% OV-17 column. The initial columntemperature was 200°C, held for 2 min and then programmedto 300C at 20°C/min. An authentic IAInos sample yielded the

characteristic four peaks of isomers with retention times of 8.1,8.7, 9.5, and 10.2 min (1 1). The plant sample had not isomerizedcompletely and yielded peaks at 8.1, 9.6, and 10.2 min. The 8.1and 8.6 peaks are pentaacetyl IAInos and the 9.6 peak is ahexaacetyl IAInos having an acetyl group on the imino nitrogen.A comparison of the mass spectral fragmentation pattern of theputative IAInos from the horse chestnut with that ofan authenticcompound isolated from Zea mays is shown in Table II.Ammonlysis of the putative IAA-myo-inositols yielded IAA

and IAA amide as determined by TLC. Gas chromatographic

1001

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I-

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w

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Table III. A Comparison of 70 eV Mass Spectral FragmentationPattern ofHeptaacetyl Rutinose Derived by Hydrolysis ofPutativeIAA-rutinosefrom A. baumanni and A. parviflora with Authentic

Sample ofHeptaacetyl Rutinose

Putative Heptaacetyl-Rutinosem/z Standard

A. baumanni A. parvifloraabundance

533

532

531

375

317

273

228

197

184

169

157

155

127

109

45

1 1

8738422449340100964073

3

5

10

6

67

62

24

43

93

38

100

98

43

76

6131711656927548737100924473

analysis on an OV- 1, 2%, 180 cm x 0.3 mm column showed anIAA peak at 9.5 min and an IAA amide peak at 12 min and a

myo-inositol peak at 6.4 min. Measurement of the GC peak areaof a sample which had been ammonolyzed with NH40H showeda stoichiometry of inositol to (IAA plus IAA amide) of 1.01 to1.00.

Isolation and Characterization of IAA-Rutinose. Endospermliquid from A. parviflora, 23 ml, was deprototeinized with 23 mlof acetone, filtered, concentrated, and chromatographed on aDowex-50 column. Tubes 16 to 35 ml contained Ehmann's-reactive material and were pooled, concentrated, and chromat-ographed on an LH-20 column. Tubes 13 to 21 containedEhmann's-reactive material and were pooled and concentrated.TLC disclosed 12 Ehmann-reactive spots as shown by the dataof Table I. The sample was concentrated and streaked out forpreparative TLC. Compounds migrating to an RlAlnml of 0.90 and0.98 were pooled and eluted from the Silica Gel with 50%ethanol. This material was concentrated and chromatographedon a C18 column using 5.5% ethanol, 0.05% acetic acid, and94.45% water. Two major peaks were obtained at retention timesof 21.6 and 26.4 min. TLC disclosed an unknown indolyliccompound at RLA.nos of 1.13, the putative IAA-rutinose at 1.07,and a mixture of IAInos and IAA-rutinose at 0.98. The 26.4peak was also an IAA-rutinose. The 21.6 min peak was concen-trated and chromatographed on a Partasil-10 column using ethylacetate:acetonitrile:ethanol:water (65:21:7:7) and yielded peaksat 3.6, 5.3, and 7.2 min. The peak at 5.3 min was concentrated,dried, dissolved in 100 ,d of 50% ethanol, and used for furthercharacterization. FAB-MS disclosed a molecular ion at 483which is detected as M+ plus a proton as is shown in Figure 2.This corresponds to an IAA ester of glucosyl-rhamnose (IAA-rutinose). The exact mass, plus 1 proton, was measured as484.1830 which agrees well with a calculated mass of 484.1819,for an empirical formula ofC22H30OI IN, . This is correct for IAA-glucosylrhamnose.A portion of the 5.3 peak was ammonolyzed using 5 zA of

sample, 5 gl of water, and 10 gl of concentrated ammoniumhydroxide. Two Ehmann-reactive spots were obtained whichcorresponded in TLC migration to IAA and indole-3-acetamide.Hydrolysis under these mild alkaline conditions indicates thatthe compound is an ester, and formation of the amide indicatesthe involvement of the IAA carboxyl in the ester linkage.A further 20 ,l fraction was ammonolyzed, dried, and acety-

lated and chromatographed isothermally at 280°C on a 6 foot

3% OV-17 column. A peak emerged with a retention time of 3.9min which corresponded exactly with that of authentic heptaacetyl rutinose. This same fraction, but prepared from A. bau-manni, was examined by GC-MS and yielded a molecular ion at533 and a fragmentation profile which corresponded exactly withthat of an authentic sample of heptaacetyl rutinose as shown bythe data of Table III.The El spectrum of the TMS derivative of the putative IAA-

rutinose is shown in Figure 3. The spectrum corresponds withIAA-1-0-glucosyl-rhamnose. After the loss of rhamnose frag-ment the ions at 653 and 450 correspond with those previouslyobserved for l-O-IAA-glucose (8). Further evidence for the lo-cation of the IAA on the 1 position of glucose is provided by thefailure of the intact IAA-rutinose to make the methoxime deriv-ative (8, 20), and by the high ratio of amide to free IAA formedby ammonlysis. The sugars would appear to be in the pyranoseconfiguration as judged by the large 204 relative to the 217 ion(6, 8, 17, 20 30). The ions 117, 129, 147, 204, 247, 291, 305,and 319 are TMS ethers of carbohydrates (7, 17, 30).

DISCUSSIONThe present work extends the known distribution of IAA-myo-

inositol since this compound had previously been described onlyfrom monocotyledonous graminae such as Zea mays and Oryzasativa (14, 19). IAInos is known to occur throughout the Zeatribe since Ehmann found it in sweet corn, field corn, popcorn,Tesosinte, and Trypsicum (6). It also occurs in vegetative tissuesof maize (4). An insufficient number of plant species have beenstudied with respect to their IAA conjugates to understanddistribution patterns and thus possibly to understand what factorsdetermine the kind of conjugates synthesized and possibly tobetter understand their function.The following conjugates have been found by methods involv-

ing extensive purification and mass spectral methods to establishthe structure of the intact compound. We specifically mentionmass spectral methods since these methods are capable of study-ing the intact molecule. This is important since other methodsmight result in a specious characterization of a mixture contain-ing A-B and C-D, and erroneously concluding that a moleculecomposed of A-C or B-D occurred.The following IAA conjugates have been found to be naturally

occurring by the above criteria: indole-3-acetyl-myo-inositol(four isomeric forms) (6, 11, 19, 27); indole-3-acetyl-myo-inosi-tol-arabinose (three isomeric forms) (6, 19, 27, 28); indole-3-acetyl-myo-inositol-galactose (three isomeric forms) (6, 27, 28);indole-3-acetyl-glucose (2, 4, and 6-0) (8, 22); [indole-3-ace-tyl]2 and 3]-myo-inositol (11); N-acetyl-indole-3-acetyl-lysine (16);indole-3-acetyl-aspartate (1, 5, 23); indole-3-acetyl-glutamate (2,12, 23, 24); indole-3-acetyl-rutinose (21, 26, and this work).Conjugates formed following the exogenous application of IAAto the plant are not included in the above list. The 1-O-IAAglucose has been shown to be formed by an enzyme from Z.mays (22) and was among the first conjugates reported (29). Abeta 1,4-IAA glucan is also known (25).The compound to which IAA is linked in ester or amide bond

is curious in that the plant does not use its most abundantconstituent for conjugation. This indicates some specificity inthe conjugating moiety. We have previously suggested that theconjugating moiety may act as a kind of zip code in determiningtransport or metabolism as has been found by Komoszynski andBandurski (18). The observation that different IAA amino acidsconjugates cause different tissue responses (15), may indicatethat, at the cellular level, the conjugating moiety causes differ-ences in the rate of hydrolysis of the conjugate or differences inits cellular location, and thus inducing a different response.Although the characterization ofthe IAA-aminodesoxy hexose

is tentative, several facts suggest this structure. There is first the

lilll



D"OMAGALSKI ET AL.

204 PYRANOSE21l.2

TMS-.OUINOUIN 1 202129 131.2\

IAA MINUS H20 3.167

24:is61.1I

200

Plant Physiol. Vol. 84, 1987

319.231.2

257. 232.2 51.2

347.2

271

6 II 1IL300

437.2

RHAMNOSE.3TMS39 .2

379..L. 1.2 42.

S1

l-O(IAA-GLUC)451.1. 220 2. 4A1.32 4A

SI .3

llL.5.31.3

lI

~ ~ W

s3;~~~~~59.4 61M.I .11111 IL

... " "-'" "- v"I.'....8.." U--.---.,, giIfl._lj I-U .11il1i IIIII 1 II I1. A.. b.400 500 600b

i-o(IAA GLUC)"3

XC16.||ttX-4 6634EX31jwliVPi7684.4 9. lR 4_ _ I.- E.4700 600

FIG. 3. 70 eV electron impactspectrum of the putative heptakisTMS IAA-rutinose isolated from A.parviflora. The molecular ion is at987. The 4-TMS l-O-IAA-glucoseOH is at 451 and the 3 TMS rham-nose is at 379. IAA ions are at 202,157, and 129. Other ions are aslabeled in the figu.

M+ IAA RUTINOSE + 7 TMSM-M.SIOH 967

915 M-TMS

4 III -IL900 1000

long retention time on Dowex-50, longer than any of the esters,but rather coincident with tryptophan. Second, there is the lowmigration on TLC despite its being an IAA monosaccharide and,last, the characteristic mass spectral fragmentation pattern. Ifourassignment is correct, this would, to our knowledge, be the firstreported occurrence of an aminodesoxoy sugar in plants.

Last, we wish to observe that greater knowledge of the kindsofIAA conjugates and their distribution in nature might provideinsight into functions of the conjugates other than those previ-ously described, such as, transport, storage, protective, and hor-monal homeostatic roles (6).

Acknowledgments.-We are indebted to Dr. Axel Ehmann for the FAB and Elspectra of the IAA-rutinose and the FAB spectrum of the putative IAA-amino-

desoxy-hexose and to Dr. Jerry Cohen for the El spectrum of the heptaacetylrutinose. The IAInos spectra were obtained at the MSU-DOE-NIH-mass spectralfacility.We gratefully acknowledge Fellowship Arrangements for W. D. by the Brethren

Service Exchange Program through the kindness of Dr. L. Gibble and ProfessorsS. W. Zagaja and S. A. Pieniazek. We acknowledge aid in manuscript preparationby Ms. Wendy Whitford and in collection and identification ofthe Horse Chestnutspecies by Professor George Parmelee.

LITERATURE CITED

1. ANDREA WA, NE GOOD 1955 The formation of indole acetyl aspartic acid inpea seedlings. Plant Physiol 30: 380-382

2. BIALEK K, JD COHEN 1986 Isolation and partial characterization of the majoramide-linked conjugate ofindole-3-acetic acid from Phaseolus vulgaris. PlantPhysiol 80: 99-104

3. CAPLAN SM, FC STEWARD 1948 Effect of coconut milk on the growth of

1112

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